Antimocrobial agent and process for producing same



1954 P. w. TANNER, JR, ETAL 2,691,618

ANTIMICROBIAL AGENT AND PROCESS FOR PRODUCING SAME 2 Sheeis-Sheet 1 Filed June 5, 1953 O WZOKUE; 2- m h ooou WKUQEDZ M O on BDNVllIWSNVHJ.

O0 o=c coon .LNBDHHd INVENTORS FRED W TANNER, .12.. JOHN E. LYNCH AND JOHN B. ROUTIEN mmae THEIR ATTORNEYS Oct. 12, 1954 F. w. TANNER, JR., ET AL 2,691,618

ANTIMICROBIAL AGENT AND PROCESS FOR PRODUCING SAME Filed June 5, 1953 2 Sheets-Sheet 2 BDNVLLIWSNVHJ. .LNHDHBd O O O O ,8 00 co v w o N- a: Q

Ill m E cn 0 Z 0'? 2 5 3 5 m 2 9 E 2 22 ag m 1: 5 00 l- :3 0:; 93 a. 2 mg O LIJ C) L 0 E 2 O o m x INVENTORS FRED W. TANNER, JR, JOHN E. LYNCH AND JOHN B. ROUTIEN THEIR ATTORNEYS Patented Oct. 12, 1954 AN TIMOCROBIAL AGENT AND PROCESS FOR PRODUCING SAME Fred W. Tanner, Jr., Baldwin, N. Y., and John E.

Lynch, East Paterson, and John B. Routien, Tenafly, N. J., assignors to Chas. Pfizer & Co.,.

Inc, Brooklyn, N.

Y., a corporation of Delaware Application June 5, 1953, Serial N 0. 359,766 8 Claims. (Cl. 167-65) This invention relates to a new and useful antibiotic called anisomycin (also known by the trade-mark Flagecidin) and more particularly, to its production by fermentation, to methods 2 with those described in Bergeys Manual of Determinative Bacteriology, sixth edition (1948). The new strain, of S. griseolus has been designated Isolate No. 14576-4 in the culture collection for its recovery and concentration from crude 5 of Chas. Pfizer & Co., Inc, of Brooklyn, N. Y. A solutions, such as fermentation broths, and to culture thereof has been deposited in the Ameriprocesses for its purification. The invention incan Type Culture Collection, Washington, D. C., eludes within its scope the antibiotic in dilute and added to its permanent collection of microforms, as crude concentrates and in pure crysorganisms as ATCC 11796. talline forms. These novel products are es- 10 The cultural characteristics of the new strain pecially useful in combatting pathogenic microof S. grz'seolus are set forth in the following table. organisms, particularly fungi and protozoa. Except where otherwise noted, the results are The new antibiotic is formed during the culbased upon six replicates incubated for two tivation under controlled conditions of a new weeks. The colors where R is written are those strain of a species of microorganism known as 5 of Ridgway, Color Standards and Nomencla- Streptomyces griseolus. The microorganism was ture.

\ Table I.-Streptomyces griseolus ATCC 11796 Color Medium Amount of growth Remarks Aerial Mycelium and Sporulation Soluble Pigment Glucose asparagine agan. Moderate Sporulation moderate, near Ben- None Creamy white reverse; spores zo Brown (R). Gram-positive, Measuring 1-1.3 (1.3) x0.65 (1.0) x1.3;4;inspira1s. Gelatin do Colonies creamy, white waxy; do Liquefaction moderate.

no sporulation, no aerial mycelium. skimmed milk (28 C.) .do Creamy white ring Coral Pink (R) No coagulation; hydrolysis; pH

V changing from 6.4 to 6.9. Glucose agar Good White 1e ernal mycelium; no Pinkish brown Reverse medium brown.

' sporu a 1011. Nutrient agar Poor Thin, flat, white aerial myce- Very light brown Reverse creamy white.

rum. Calcium malate agar Poor to moderate White aerial mycelium; no None Calcium malate digested; reverse sporulation. creamy White. Synthetic agar Poor Poor sporulation, near Pale .....do Reverse creamy white to light Mouse Gray (R). gray. Emersons agar Moderate Moderate sporulation, near Light brown Reverse light brown.

Mouse Gray (R). Cellulose None Dextrose nitrate broth. N 0 reduction of nitrates. Potato discs Sporulation darker than Mouse Gray (R). Starch agar Moderate Sporulation near Benzo Brown Yellow subsurface growth; re- (R). verse light brown, narrow zone of hydrolysis.

identified by planting and testing cultures thereof on media normally used for such identification, and comparing the cultural characteristics Our new strain of S. griseolus differs from the described strains thereof in several ways which are enumerated in the following table.

Table II Medium S. griseolus ATCC 11796 Bergeys Description Gelatin Q. Colonies creamy, white waxy (in Petri dishes) Yellowish, flaky pellicle and sediment (in gelatin stabs). Synthetic agar Poor Sporulation, near Mouse Gray (R); growth poor. Aerial mycelium at first gray, later becoming pallid,

Glucose agar Litmus milk white ring; Coral Dextrose nitrate broth.

Good growth; white aerial mycelium; no sporulatiou (On skimmed milk at 28 C.) Moderate growth; creamy Pink (R) soluble pigment; no coagulation; hydrolysis; pH becomes alkaline.

N 0 reduction to nitrites dar Abundant growth, pink pellicle; coagulated; peptonized;

becoming alkaline.

N itrltes produced.

It is to be understood that for the production of anisomycin the present invention is not limited to the aforesaid organism or to organisms fully answering the above description, which is given only for illustrative purposes. In fact, it is especially desired and intended to include the use of mutants produced from the described organism by various means, such as X-radiation, ultraviolet radiation, nitrogen mustards, and the like.

While anisomycin shows activity against a variety of microorganisms, as previously mentioned, it is particularly active against pathogenic protozoa and fungi. The following table illustrates the antibiotic spectrum of anisomycin through tests performed against a variety of such fungi. These tests were run by seeding nutrient broth containing various concentra-' tions of the pure antibiotic with the particular organism specified and observing the concentrations at which no growth and growth, respectively, occurred. The antibiotic was employed in concentrations of 1, 10, 100 and 500 micrograms/milliliter (meg/ml). The test was conducted under standardized conditions.

Table III .-Spectrum of anisomycin, pathogenic Sporotrichum schenkiz' Trichophyton violaceum Hormo'dendron compactum. Blastomyces brasiliensis The antifungal activity of anisomycin is further illustrated in Table IV, which shows the minimum concentration of antibiotic at which growth of various strains of Candida albicans failed to occur. This concentration is expressed as the minimum inhibitory concentration in meg/ml. A standardized procedure was employed, similar to that described above with respect to Table III. The antifungal effectiveness of anisomycin in humans has not as yet been demonstrated.

Table IV.-Spectrum of anisomycin, Candida albicans Minimum Inhibitory Concentra- Candida Albicans Strain No. on Amy) Pl indicates partial inhibition.

The antiprotozoan activity of anisomycin was determined by innoculating tubes containing various concentrations of the antibiotic, with typical pathogenic protozoa, Trichomonas oagz'nalis and Endamoeba histolytica, and observing the degree of motility and/or growth of the microorganisms after incubation for 48 hours. For purposes of comparison, the same tests were employed with the antibiotic, fumagillin, which is known for its antiprotozoan activity. The results of these tests appear in Table V, wherein the ratings for activity against Trichomonas are based on the motility and growth observed in slides prepared from tubes incubated at the concentrations specified. In rating activity against Trichomonas, a figure of 4+ indicates complet inhibitory activity of the antibiotics tested, 1. e., neither motility nor growth of the microorganism; 3+ indicates marked activity of the antibiotic, or slight motility and/or growth; 2+ indicates moderate activity; 1+ indicates slight activity; and indicates no activity. Activity against Endamoeba was determined by comparing the number of cells in control tubes with the number of cells in tubes containing the antibiotics in the concentrations specified, th tubes being rated for complete inhibitory activity and for no activity.

Table V.--Anti-protozoan activity of anisomycin Activity at (floncentrations o Antibiotic Protozoa 7.8 mcg./ 3.9 meg. 1.9 mag] ml. ml. ml.

Triehomonas 4+ 3+ 2+ Endamoeba-.- Fumagillin Trichomonaa. 3+ Fumagillin Endamoebam 1 No activity was observed below 15.6 meg/1n].

. organisms.

Anisomycin has been found to possess a relatively low level of toxicity when used in test animals. For example, the LDo value, when the antibiotic is administered intravenously to mice as a solution in water, is approximately 2 mg./20- gram. mouse.

The invention includes within its scope processes for growing the microorganism S. griseoius ATCC 11796. The cultivation of the microorganism preferably takes place in aqueous nutrient media at a temperature of about 24-30 C., and under submerged conditions of agitation and aeration. Nutrient media which are useful for these processes include a carbohydrate, such as sugars, starch, glycerol, corn meal, and a source of organic nitrogen, such as casein, soybean meal, peanut meal, wheat gluten, cottonseed meal, lactalbumin, enzymatic digest of casein, tryptone. A source of growth substances, such as distillers solubles, yeast extract, molasses fermentation residues, as well as mineral salts, such as sodium chloride, potassium phosphate, sodium nitrate,

magnesium sulfat and trace minerals such as copper, zinc and iron may also be utilizedwith desirable results. If excessive foaming is encountered during the fermentation, anti-foaming agents, such as vegetable oils,,may be added to the fermentation medium. The pH of thefermentation tends to remain rather constant, but, if variations are encountered, a buffering agent, such as calcium carbonate may also .be added to the medium.

Inoculum for the preparation of anisomycin by the growth of S. grz'seolus may be obtained by employing growth from slants on such media as Emersons agar or beef lactose. The growtli may be used to inoculate either shaken flasks or inoculum tanks for submerged growth, or, alternatively, the inoculum tanks'may be seeded from the shaken flasks. The growth of the microorganism usually reaches its maximum in about two or three days. However, variations in the equipment used, the rate of aeration, rate of stirring and so forth may affect the speed with which the maximum activity is reached. In general, the fermentation is continued until substantial antimicrobial activity is impartedto themedium, a period from about 24 hours to four days being sufficient for most purposes. Aeration of the medium in tanks for submerged growth is maintained at the rate of about onehalf to two volumes of free air per volume of broth per minute. Agitation may be maintained by suitable types of agitators generally familiar to those in the fermentation industry. Aseptic conditions must, of course, be maintained throughout the transfer of the inoculum and throughout the growth of the microorganism.

After growth of the microorganism, the mycelium, which is generally quite luxuriant and fine, may be removed from the fermentation broth by various standard equipment, such as filterpresses, centrifuges, and so forth. Thereafter, the antibiotic may be recovered from the fermentation broth by several different procedures. Alternatively, th whole broth may be used as is or it may be dried. The antibiotic may be further purified by various means; for instance, the compound may be extracted from aqueous solution at neutral or slightly alkaline pHs, preferably between about 6 and about 10, by means of a variety of water-immiscible organic solvents, including ethers, aromatic hydrocarbons, esters, ketones, lower alcohols, halogenated hydrocarbons, and mixtures thereof. Examples of these are diethyl ether, benzene, ethyl acetate, butyl acetate, methyl isobutyl ketone, butanol, and

chloroform. The antibiotic may be extracted from most solvent solutions back into acidified water, preferably at a pH of below about 2.5. If desired, the solvent extract may be concentrated before extraction into acidified water. By adjustment of the pH to neutrality 0r alkalinity, the antibiotic may be re-extracted into one of the solvents indicated above. Upon drying the solvent and concentrating the solution, the antibiotic crystallizes in long whiteneedles. The

product may be recrystallized by cooling a solution thereof in hot butanol, ethyl acetate, benzene, or ethylene dichloride. Other -methods of recovery which suggest themselves include absorption on charcoal with subsequent elution, treatment with ion exchange resins, and development on alumina columns.

Anisomycin is a basic, white organic compound that is soluble in dilute, aqueous acids, and moderately soluble in water. It is very soluble in a number of organic solvents, such as methyl alunder anhydrous conditions.

- cohol, ethyl alcohol, acetone, dioxane and chloroand 283.5 m respectively (3.34 mg. in 25 ml.

methanol). When dissolved in chloroform, the antibiotic shows a number of characteristic peaks in the infrared region, the more significantof which are at the following frequencies (in reciprocal centimeters): 3545, 3450, 3320, 2890, 2800, 1725, 1610, 1582, 1515, 1470, 1447, 1380, 1320, 1302, 1242, 1178, 1036, and 962. The infrared spectrum is more particularly illustrated in the attached drawings, wherein Fig. 1 shows the spectrum at frequencies from 5000 to 1100 emf and Fig. 1a from 1100 to 625 cmr Thebase dissolved in methanol (C, 1%) and allowed to come to equilibrium has an [a] :30.0. When dissolved in chloroform (C, 1%), its optical rotation is [a] =45i3.

A sample of anisomycin, which had been crystallizedfrom ethyl acetate, was dried for three hours at 56 0. without loss in weight. The dried, crystalline antibiotic was then analyzed and found to be composed of the following elements in the proportions by weightspecified:

Carbon 63.55 Hydrogen -1 7.27 Nitrogen 5.20 Oxygen (by difference) 23.98

This corresponds to the probable empirical formula C14H19NO4 for the basic antibiotic.

Anisomycin is clearly distinguished fromother antibiotics by its properties, as evidenced by those properties described above and by paper chromatography measurements. Useful salts of the antibiotic can be prepared by methods well known in the art, as by treatment of the base with theappropriate acid in aqueous solution or For instance, the hydrochloride can be prepared by dissolving the base in acetone and passing hydrogen chloride gas into the solution. Other acids, such as sulfuric, phosphoric acids, may be used to make the acid salts of the antibiotic.

The invention is further illustrated by the following example, which is not to be considered as imposing any limitation thereon.

Errample lowing materials in one liter of water:

Grams Glucose (Cerelose) 10 Corn starch 10 Hydrolyzed casein (N-Z Amine B) 5 Distillers molasses solubles (Curbay BG) 5 Soybean meal 15 Sodium chloride 1 After adjusting the mixture to a pH of 7 with potassium hydroxide, one gram of calcium carbonate was added, and steam was passed through the mixture for about 30 to 45 minutes to sterilize the same. A slant culture of the new strain of .S'. griseolus was transferred to m1. of this medium in a 300 m1. Erlenmeyer flask and shaken 45 hoursuntil good growth was obtained. Inocuslum for a larger fermentation was prepared by transferring the contents of the aforesaid flask under aseptic conditions to one liter of the same medium in a 3 liter flask and shaking the same for 48 hours.

Fifty gallons of nutrient medium were prepared and sterilized as above indicated and 'innoculated with the inoculum thus prepared. The organism was then cultivated under submerged conditions vof aeration and agitation tor .a period of three days. The fermentation broth was adjusted to a pH of about 2 with concentrated sulfuric .acid. and filtered with Supercel to remove the myocl-ium. Next, the filtrate broth was adjusted to pH 9 with concentrated sodium hydroxide and twice extracted with 15 gallons of chloroform.

The combined chloroform extracts were concentrated and extracted with water acidified to pH 2 with concentrated sulfuric acid. Thereafter, the acidified aqueous solution was concentrated andreadjusted to p119 with further sodium hydroxide. The antibiotic was .re-extrwted into- -ch1oroform which was evaporated to about a volume of .50 ml. Upon the addition of an equal volume of cyclohexane and further evaporation, the crude antibiotic was crystallized sand recovered by filtration. The crude antibiotic was purihad by dissolving the same in ethyl acetate, treating the resulting solution with absorbent carbon, and evaporating the ethyl acetate to obtain lon white crystals of .anisomycin.

Resort may be had to such modifications and equivalents .as fall within the spirit of the invention and the scope of the appended claims.

What we .claim ,1. A process for producing .anisomycin, which comprises cultivating the microorganism Streptomyces griseo'lus ATCC 11796 than aqueous nutrient medium under submerged aerobic conditions until substantial antimicrobial activity is impar-told to said medium.

2. A process as claimed in claim 1 wherein the anisomycin -is recovered from the termenta'tlon broth.

3. A process as claimed in claim 1 wherein .anisomycin is recovered 'zfrom the fermentation broth by filtering the broth and extracting it with a water-immiscible organic solvent selected tnom the group consisting :of ethers, aromatic hydrocarbons, esters, 'lcetones, lower alcohols, halogenated hydrocarbons and mixtures thereof, :under neutral to alkaline pH conditions.

4. A process for producing .anisomycin, which comprises cultivating Streptomyces griseolus ATCC 11796 in an aqueous nutrient medium under agitated submerged aerobic conditions at a temperature of from about 24 to about 30 C., .for a period of from about one day to aboutjour days.

5. A substance effective in inhibiting thegrowth of pathogenic protozoa and fungi, selected from the group consisting of a basic substance moderately soluble in water, very soluble in methyl .211- cohol, ethyl alcohol, acetone, dioxane and chloroform, insoluble in hexane, cyclohexane, carbon tetrachloride and ether, and capable of forming salts with acids; whose crystalline 'base contains the elements carbon, hydrogen, nitrogen and oilyteen in substantially the fol-lowing proportions by whose crystalline base displays three peaks, at 224, .277 and 283.5 m respectively, in the ultraviolet region of the spectrum, when dissolved in chl roform .(C, 1%) has the optical rotation [-oc =-=5j:3, and when dissolved in chloroform, exhibits characteristic absorption in the infrared region at the following frequencies expressed in reciprocal centimeters: 3545, 3450, 3320, 2.890, 280.0, 1725,1610, 1582, 1515, 1470,1460, 1447, 1380, 1320, 130.2, 1.242, 1178., 10,36 and 962; and the acid salts of said basic substance.

6. A basic substance effective in inhibiting the growth of pathogenic protozoa and fungi and capable of iorming salts with acids; which basic substance is moderately soluble in water, very soluble in methyl alcohol, ethyl alcohol, acetone, dioxane .and chloroform, insoluble in hexane, cyc'lo'hexane, carbon tetrachloride and ether, whose crystalline base contains the elements carbon, hydrogen, mtrogen, and oxygen in substantially the iollowing proportions by weight:

Carbon 6.3.55 Hydrogen 7:27 Nitrogen 5:20 Oxygen -.(.by difference) 23.98

whose crystalline base displays three peaks at 22.4, .277 and 283.5 ml, respectively, in the ultraviolet region of the spectrum, when dissolved in chloroform .(C, 1%) has the optical rotation [al =-.4.5:3, and when dissolved in chloroior'm, exhibits characteristic absorption ,in the infrared region at the following frequencies expressed in reciprocal centimeters: .3545, .3450, .3320, 289.0, 2800, 1725,1610, 1582, 1515, 147.0, 1460, .1447, 138.0, .1320, 1.302, 1242, 1178, 1.036 and .962.

7. .Anacidsalt of the basicsubstance defined in .claim .6.

8. .A hydrochloride .oi the basic substance .defined in claim 6.

I'Beferences Cited in the file of this patent UNITED STATES PATENTS Number Name Date 2,378,876 Waksman June 19, "1945 2,482,055 Duggar Sept. 13, 1949 2,516,080 Sobi-n et a1. July 18, 1950 2617;755 Benedict et a1. Nov. '11, 1952 2,549,401 "H-aines etal. Aug. '8, 1953 2,653,899 'Bunch'etial Sept. 29, 1953 =OT-HER REFERENCES Waksman: The Actinomycetes, .pub. by the ChronicaBotanica (10., Waltham, Mass, 1950, pp. 116-117..

Seneca .et .al. Antibiotics and Chemotherapy," July 1952 .pp. 357-4360.

Hewitt -.et al..: Antibiotics and Chemotherapy. August 1.9.52, vol. 2, No. =8, pp. 409-410.

De Somer .et,al.: Antibiotics and Chemothera- ,py, .May 1954, pp. 546-550. 

1. A PROCESS FOR PRODUCING ANISOMYCIN, WHICH COMPRISES CULTIVATING THE MICROORGANISM STREPTOMYCES GRISEOLUS ATCC 11796 IN AN AQUEOUS NUTRIENT MEDIUM UNDER SUBMERGED AEROBIC CONDITIONS UNTIL SUBSTANTIAL ANTIMICROBIAL ACTIVITY IS IMPARTED TO SAID MEDIUM.
 5. A SUBSTANCE EFFECTIVE IN INHIBITING THE GROWTH OF PATHOGENIC PROTOZOA AND FUNGI, SELECTED FROM THE GROUP CONSISTING OF A BASIC SUBSTANCE MODERATELY SOLUBLE IN WATER, VERY SOLUBLE IN METHYL ALCOHOL, EHTYL ALCOHOL, ACETONE, DIOXANE AND CHLOROFORM, INSOLUBLE IN HEXANE, CYCLOHEXANE, CARBON TETRACHLORIDE AND ETHER, AND CAPABLE OF FORMING SALTS WITH ACIDS; WHOSE CRYSTALLINE BASE CONTAINS THE ELEMENTS CARBON, HYDROGEN, NITROGEN AND OXYGEN IN SUBSTANTIALLY THE FOLLOWING PROPORTIONS BY WEIGHT: 